Array hybridization method including determination of completeness of restriction digest

Abstract

The present invention provides a method of performing an array hybridization analysis of a sample, including performing a restriction digest reaction on the sample, hybridizing the digested sample to the array, and interrogating the array. The array includes probe sets that provide for a determination of the extent of the restriction digest performed on the sample. Arrays including the probe sets are also described.

1 . A method of performing an array hybridization analysis of a sample, the method comprising: 
 a) performing a restriction digest reaction on the sample to yield a digested sample,    b) hybridizing the digested sample to an array, wherein the array comprises a probe series, the probe series comprising at least one probe set, each of the at least one probe sets comprising a junction probe and a cognate flanking probe,    c) interrogating the array to obtain a junction hybridization signal and a cognate flanking hybridization signal, and    d) comparing the junction hybridization signal and cognate flanking hybridization signal to determine the extent of the restriction digest reaction.    
   
   
       2 . The method of  claim 1 , wherein the probe series comprises at least  5  probe sets, wherein interrogating the array provides a junction hybridization signal and a cognate flanking hybridization signal for each of the at least 5 probe sets, and wherein comparing comprises comparing the junction hybridization signal and cognate flanking hybridization signal for each of the at least 5 probe sets to determine the extent of the restriction digest reaction.  
   
   
       3 . The method of  claim 2 , wherein comparing includes discarding one or more outlying junction hybridization signals and the respective cognate flanking signals and determining the extent of the restriction digest reaction with the remaining junction hybridization signals and cognate flanking hybridization signals.  
   
   
       4 . The method of  claim 2 , wherein comparing includes weighting one or more outlying junction hybridization signals and the respective cognate flanking signals less than the remaining junction hybridization signals and cognate flanking hybridization signals.  
   
   
       5 . The method of  claim 1 , wherein interrogating comprises illuminating the array and detecting the location and intensity of resulting fluorescence at multiple features of the array.  
   
   
       6 . The method of  claim 1 , wherein the restriction digest reaction comprises contacting the sample with at least two restriction endonucleases, and the array comprises a probe series for each of the at least two restriction endonucleases.  
   
   
       7 . The method of  claim 6 , wherein each probe series comprises at least one probe set, each probe set comprising a junction probe and at least one flanking probe.  
   
   
       8 . The method of  claim 1 , wherein the junction probe of each of the at least one probe sets has a bridge site at a position between about 30% and about 70% of the distance along the junction probe.  
   
   
       9 . The method of  claim 1 , wherein the array further comprises primary probes directed to known sequences of genomic template, wherein the array does not include junction probes directed to sequences within about 1000 bases (600, 500, 400, 300) from the known sequences of genomic template that the primary probes are directed to.  
   
   
       10 . The method of  claim 9 , wherein the number of primary probes on the array is at least about 5 times the total number of junction probes on the array and is less than about 5000 times the total number of junction probes on the array.  
   
   
       11 . The method of  claim 9 , wherein the number of primary probes on the array is at least about 5 times the total number of junction probes on the array and is less than about 100 times the total number of junction probes on the array.  
   
   
       12 . The method of  claim 9 , wherein the number of primary probes on the array is at least about 100 times the total number of junction probes on the array and is less than about 5000 times the total number of junction probes on the array.  
   
   
       13 . The method of  claim 1 , wherein the array hybridization analysis is adapted to provide a measure of copy number variation in the sample.  
   
   
       14 . The method of  claim 1 , wherein the sample includes reference target and analyte target, wherein the reference target and the analyte target are differentially labeled.  
   
   
       15 . The method of  claim 1 , wherein hybridizing is performed under stringent conditions.  
   
   
       16 . The method of  claim 1 , wherein the array comprises at least 5 probe sets.  
   
   
       17 . The method of  claim 16 , wherein each probe set comprises at least two cognate flanking probes for each junction probe.  
   
   
       18 . The method of  claim 17 , wherein the least two cognate flanking probes for each junction probe include an upstream flanking probe and a downstream flanking probe.  
   
   
       19 . The method of  claim 1 , wherein the junction probe of each probe sets bridges a restriction site of a known template sequence and is complementary to the know template sequences immediately adjacent the restriction site, wherein the restriction digest reaction can cut at the restriction site, further wherein the cognate flanking probe of each probe set is directed to a portion of the known template sequence that is within about 1000 bases of the restriction site of the known template sequence.  
   
   
       20 . The method of  claim 19 , wherein the cognate flanking probe of each probe set is directed to a portion of the known template sequence that is adjacent the restriction site of the known template sequence.  
   
   
       21 . The method of  claim 1 , wherein the method is performed in conjunction with an array CGH assay.  
   
   
       22 . An array comprising a first probe series, the first probe series comprising a plurality of probe sets, each of the plurality of probe sets comprising a junction probe and at least one flanking probe, each of the plurality of probe sets directed to a different restriction site.  
   
   
       23 . The array of  claim 22 , wherein the at least one flanking probe of each probe set is directed to a sequence that is within about 1000 bases from the restriction site that the probe set is directed to.  
   
   
       24 . The array of  claim 22 , wherein the at least one flanking probe of each probe set comprises at least one upstream flanking probe and at least one downstream flanking probe.  
   
   
       25 . The array of  claim 22 , wherein at least one of the at least one flanking probes of each probe set is directed to a sequence directly adjacent the restriction site that the probe set is directed to.  
   
   
       26 . The array of  claim 22 , wherein at least one of the at least one flanking probes of each probe set overlaps the at least one junction probe from the same probe set.  
   
   
       27 . The array of  claim 22 , wherein in certain embodiments, the calculated melting temperatures of at least about 80% of the flanking probes and the junction probes on an array fall within a range of about 6 degrees Celsius.  
   
   
       28 . The array of  claim 22 , wherein the array further comprises a second probe series, the second probe series comprising a plurality of probe sets, each of the plurality of probe sets of the second probe series comprising a junction probe and at least one flanking probe, each of the plurality of probe sets of the second probe series directed to a different restriction site which may be cleaved by a second restriction endonuclease, and the probe sets of the first probe series are directed to restriction sites which may be cleaved by a first restriction endonuclease which is different from the second restriction endonuclease.